1. Field of the Invention
The present invention concerns an improved method for the isolation of nucleic acids.
2. Description of Related Art
The isolation of nucleic acids such as DNA and RNA from plant, animal or human cells as well as from cell cultures or virus cultures is normally carried out according to a uniform basic pattern: the starting materials containing the nucleic acids are first digested—in part with the use of protein-degrading enzymes. The individual components can then be separated in subsequent steps by many different methods.
The separation of the protein fraction inevitably present in every cell lysate embodies here a particularly important step. The separation can be carried out, for example, by bringing the protein/nucleic acid mixture into contact with phenol and/or mixtures of chloroform/isoamyl alcohol. The protein fraction can also be precipitated from the aqueous phase by the addition of denaturing salts—such as, for example, guanidinium hydrochloride or guanidinium isothiocyanate. In addition the proteins can be degraded by the addition of proteases and then removed. Finally the unwanted nucleic acid can be separated by the selective addition of DNase or RNase and the respectively desired nucleic acid fraction can be obtained. However, to protect the nucleic acids from unwanted enzymatic degradation during the isolation procedure, work must be carried out under sterile and nuclease-free conditions. The separation of nucleic acids can also be carried out by ultracentrifugation.
Most of the methods known from the prior art are based on one of the following two separation principles:
The “classical methods” are based on a single stage process in which after addition of a buffer, which in most cases contains a guanidinium salt, and after addition of an organic extraction agent—mostly chloroform or phenol—an extraction is carried out. The undesirable attendant materials are then rejected with the organic phase. The nucleic acids remaining in the aqueous phase can then be separated by a phase separation and isolated.
The main disadvantage of this method is that in addition to the use of toxic and health-hazardous materials—such as guanidinium isothiocyanate, phenol or chloroform, water-soluble materials remain in the aqueous nucleic acid solution as impurities, which must be separated in additional, very time-consuming purification steps. This problem complicates the use of this method for the isolation of nucleic acids from plants, for example, since these mostly contain considerable amounts of polysaccharides and similar water-soluble substances.
In view of these disadvantages an alternative method has become established in the prior art which is based on the selective adsorption of nucleic acids onto solid, usually mineral, carriers such a silicon dioxide. Here, in a multi-stage procedure different buffer solutions (lysis, binding, washing and elution buffers) are added sequentially to the cell or virus lysate; in the final step the purified nucleic acid is eluted from the carrier.
Meanwhile expert circles have investigated the physico-chemical principle of the binding of nucleic acids to mineral carriers in the presence of chaotropic salts. It was postulated that the binding of the nucleic acid to the surface of the mineral carrier is based on a disruption of the highly ordered structures of the aqueous milieu, through which the nucleic acids adsorb onto the surface of mineral materials, in particular glass and silica particles.
A particular disadvantage of the above-described method is that with the use of samples that are enhanced with a particularly high fraction of spurious secondary materials, considerable losses in yields must be taken into account to achieve the desired high level of purity.